1. Volume 104, Issue 4, Pages 934-942 (February 2013)
Stochastic Model-Assisted Development of Efficient Low-Dose Viral Transduction in Microfluidics 
Camilla Luni, Federica Michielin, Luisa Barzon, Vincenza Calabrò, Nicola Elvassore 
Biophysical Journal 
Volume 104, Issue 4, Pages (February 2013)
DOI: /j.bpj
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2. Figure 1
Microfluidic experimental setup. (A) Three-dimensional graphical representation of the microfluidic platform composed of 10 parallel independent channels. (B) Lateral section of one channel indicating its geometrical dimensions. (White arrow) Flow direction. (C) Overview of the whole system: the microfluidic platform (top view on the left) is placed in a biological incubator during experiments, and medium perfusion (from left to right) in every channel is provided by a set of syringe pumps, whose temporal pattern of flow rate is automatically controlled. (D) Images of the whole microfluidic channel taken with a fluorescence microscope to detect EGFP+ cells and cell nuclei.
Biophysical Journal  , DOI: ( /j.bpj )
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3. Figure 2
Stochastic model processes and parameter fitting. (A) Graphical representation of the phenomena included in the stochastic model: medium convection with parabolic velocity profile, virus Brownian motion in three-dimensional space, and virus entering a cell with a certain probability when it gets on its surface. Plane x-z represents the bottom surface of the channel where cells are randomly distributed within a regular square grid. (B) Results of model parameter fitting. Experimentally, transduction was performed in a 24-well plate using 200 μL of virus-containing medium for 90 min. Cell concentration was 130 cell/mm2. (Red error bars) Percentage of cells expressing EGFP 24 h after AdV transduction as a function of virus concentration, mean ± standard deviation values obtained in independent experiments, each performed in double or triple. Simulations by the stochastic model reproduce the experimental conditions. In the model, EGFP+ cells are given by cells infected by at least one virus. (Black dots) Model outcome, each dot is obtained from one simulation at the given virus concentration. One-hundred simulations were performed at each condition. (Black line) Simulation mean results.
Biophysical Journal  , DOI: ( /j.bpj )
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4. Figure 3
Computational study of spatial heterogeneity in the microfluidic system. (A and C) Results of stochastic simulations of continuous channel perfusion with a flow rate of 0.1 μL/min for 90 min. (B and D) Results of simulations of discontinuous perfusion: 2 min of inflow at 6 μL/min, 90 min without perfusion, and 2 min of outflow at 6 μL/min. (A and B) Bottom of a microfluidic channel: the EGFP+ cells (top) and the number of virus/cell (bottom) are shown; color meaning is explained in color bars in panel A. (Black arrow) Flow direction. (C and D) (Dotted lines) Percentage of EGFP+ cells in each of the 10 equal sectors of the channel. (Error bars) Mean ± standard deviation of 100 simulations. (A–D) Cell concentration is 130 cell/mm2, instantaneous MOI is 20.
Biophysical Journal  , DOI: ( /j.bpj )
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5. Figure 4
Computational study of viral transduction at different MOIs. Results of 100 stochastic simulations at MOI 1 (A), 5 (B), 10 (C), and 20 (D), in a microfluidic channel under discontinuous flow conditions, at a cell concentration of 150 cell/mm2. Bar plots represent the percentage of EGFP+ cells infected by n viruses. (Solid lines) Complementary cumulative distribution. In each plot the percentage of EGFP+ cells (i.e., cells infected by at least one virus) is also indicated. (Error bars) Mean ± standard deviation of the 100 simulations.
Biophysical Journal  , DOI: ( /j.bpj )
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6. Figure 5
Experimental study of viral transduction in the microfluidic platform at different MOIs. (A) Comparison of experimental (subscript exp) and simulated (subscript mod) percentage of EGFP+ cells. Marker colors are defined by legend in panel B. (B) Complementary cumulative distribution of the percentage of EGFP+ cells showing a fluorescence intensity > f (see Cell Characterization in main text). (C) Probability distribution of the percentage of EGFP+ cells showing a fluorescence intensity f at MOI 1, 5, 10, and 20 (from left to right). (A–C) The percentage of cells expressing EGFP was detected 24 h after AdV transduction. Cell concentration was 150 cell/mm2. (Error bars) Mean ± standard deviation values obtained in independent experiments, each performed in double or triple.
Biophysical Journal  , DOI: ( /j.bpj )
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7. Figure 6
Multiple viral transductions strategy and computational results. Repeated 90-min AdV infections with MOI 20 (A), 10 (B), and 5 (C), at the time-points indicated. (Insets) Model predictions of the percentage of EGFP+ cells infected by n viruses at the end of each pulse of infection. Cell concentration was 150 cell/mm2. (Error bars) Mean ± standard deviation of 100 simulations at each condition.
Biophysical Journal  , DOI: ( /j.bpj )
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8. Figure 7
Results from experiments of multiple viral transductions within the microfluidic platform. AdV transduction timing followed the strategy shown in Fig. 6: one infection at MOI 20 (A and blue line in D), two infections at MOI 10 (B and red line in D), and 4 at MOI 5 (C and green line in D). EGFP+ cell fluorescence intensity, f, was measured at 24, 36, 48, and 60 h after the first infection. Cell concentration was 150 cell/mm2. (Error bars) Mean ± standard deviation of experiments repeated twice. (A–C) Probability distribution of the percentage of EGFP+ cells showing a fluorescence intensity f, and (D) associated complementary cumulative distribution.
Biophysical Journal  , DOI: ( /j.bpj )
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